A current focus of interest in molecular biology and biotechnology is in the display of large libraries of proteins and peptides and in means of searching them by affinity selection. The key to genetic exploitation of a selection method is a physical link between individual molecules of the library (phenotype) and the genetic information encoding them (genotype). A number of cell-based methods are available, such as on the surfaces of phages (1), bacteria (2) and animal viruses (3). Of these, the most widely used is phage display, in which proteins or peptides are expressed individually on the surface of phage as fusions to a coat protein, while the same phage particle carries the DNA encoding the protein or peptide. Selection of the phage is achieved through a specific binding reaction involving recognition of the protein or peptide, enabling the particular phage to be isolated and cloned and the DNA for the protein or peptide to be recovered and propagated or expressed.
A particularly desirable application of display technology is the selection of antibody combining sites from combinatorial libraries (4). Screening for high affinity antibodies to specific antigens has been widely carried out by phage display of antibody fragments (4). Combinations of the variable (V) regions of heavy (H) and light (L) chains are displayed on the phage surface and recombinant phage are selected by binding to immobilised antigen. Single-chain (sc) Fv fragments, in which the VH and VL domains are linked by a flexible linker peptide, have been widely used to construct such libraries. Another type of single chain antibody fragment is termed VH/K, in which the VH domain is linked to the complete light chain, i.e. VH-linker-VL-CL (10). This has a number of advantages, including stability of expression in E. coli and the use of the CL domain as a spacer and as a tag in detection systems such as ELISA and Western blotting. Antibody VH and VL region genes are readily obtained by PCR and can be recombined at random to produce large libraries of fragments (21). Such libraries may be obtained from normal or immune B lymphocytes of any mammalian species or constructed artificially from cloned gene fragments with synthetic H-CDR3 regions (third complementarity determining region of the heavy chain) generated in vitro (22). Single chain antibody libraries are potentially of a size of  greater than 1010 members. Libraries can also be generated by mutagenesis of cloned DNA fragments encoding specific VH/VL combinations and screened for mutants having improved properties of affinity or specificity. Mutagenesis is carried out preferably on the CDR regions, and particularly on the highly variable H-CDR3, where the potential number of variants which could be constructed from a region of 10 amino acids is 2010 or 1013.
It is clear that for efficient antibody display it is necessary to have a means of producing and selecting from very large libraries. However, the size of the libraries which can potentially be produced exceeds by several orders of magnitude the ability of current technologies to display all the members. Thus, the generation of phage display libraries requires bacterial transformation with DNA, but the low efficiency of DNA uptake by bacteria means that a typical number of transformants which can be obtained is only 107-109 per transformation. While large phage display repertoires can be created (17), they require many repeated electroporations since transformation cannot be scaled up, making the process tedious or impractical. In addition to the limitations of transformation there are additional factors which reduce library diversity generated with bacteria, e.g. certain antibody fragments may not be secreted, may be proteolysed or form inclusion bodies, leading to the absence of such binding sites from the final library. These considerations apply to all cell-based methods. Thus for libraries with 1010 or more members, only a small fraction of the potential library can be displayed and screened using current methodologies. As noted, the size of an antibody library generated either from animal or human B cells or artificially constructed can readily exceed 1010 members, while the number of possible peptide sequences encoding a 10 residue sequence is 1013.
In order to avoid these limitations, alternative display systems have been sought, in particular in vitro methods which avoid the problem of transformation in library production. One such method is the display of proteins or peptides in nascent form on the surface of ribosomes, such that a stable complex with the encoding mRNA is also formed; the complexes are selected with a ligand for the protein or peptide and the genetic information obtained by reverse transcription of the isolated mRNA. This is known as ribosome or polysome display. A description of such a method is to be found in two U.S. patents, granted to G. Kawasaki/Optein Inc. (16). Therein, semi-random nucleotide sequences (as in a library) are attached to an xe2x80x98expression unitxe2x80x99 and transcribed in vitro; the resulting mRNAs are translated in vitro such that polysomes are produced; polysomes are selected by binding to a substance of interest and then disrupted; the released mRNA is recovered and used to construct cDNA. Two critical parts of the method are the stalling of the ribosome to produce stable complexes, for which cycloheximide is used, and the recovery of the mRNA, for which the bound polysomes are disrupted to release mRNA and the mRNA is then separately recovered. The latter is an integral part of the method as described by Kawasaki and adopted by all others until now. Thus, section VII of the patents (16) deals with the disruption of the polysomes by removal of magnesium, etc; no other method for recovery of RNA or cDNA is suggested other than ribosomal disruption. In U.S. Pat. No. 5,643,768, claim 1 refers to translating mRNA in such a way as to maintain polysomes with polypeptide chains attached, then contacting to a substance of interest, and finally isolating mRNA from the polysomes of interest. In claim 2, cDNA is constructed subsequent to isolating mRNA from the polysomes that specifically bind to the substance of interest. This is reiterated in claim 15, wherein step (g) comprises disrupting said polysomes to release said mRNA and step (h) comprises recovering said mRNA, thereby isolating a nucleotide sequence which encodes a polypeptide of interest. Similarly, this is repeated again in claim 29 (e) . . . isolating mRNA from the polysomes that specifically react with the substance of interest. In U.S. Pat. No. 5,658,754, claim 1 (g) also requires disrupting said polysomes to release mRNA; (h) is recovering said mRNA; and (i) is constructing cDNA from said recovered mRNA. However, Kawasaki did not reduce the method to practice in these filings and provided no results. Accordingly, the method was not optimised and he was unaware of the inefficiency of the system as he described it, in particular that due to the method of recovery of mRNA by polysome disruption.
Another description of prokaryotic polysome display, this time reduced to practice, is the international published application WO 95/11922 by Affymax Technologies (18) and the associated publication of Mattheakis et al. (14). Both relate to affinity screening of polysomes displaying nascent peptides, while the patent filing also claims screening of antibody libraries similarly displayed on polysomes. They refer to libraries of polysomes, specifically generated in the E. coli S30 system in which transcription and translation are coupled. To produce a population of stalled polysomes, agents such as rifampicin or chloramphenicol, which block prokaryotic translation, are added. The means of recovering the genetic information following selection of stalled ribosomes is again by elution of the mRNA. In the flowsheet of the method shown in FIG. 10 of the patent application (18), an integral part is step 4, namely elution of mRNA from the ribosome complexes prior to cDNA synthesis. The main example in the patent and the publication is of screening a large peptide library with 1012 members by polysome display and selection of epitopes by a specific antibody. The polysomes were selected in antibody-coated microplate wells. The bound mRNA was liberated with an elution buffer containing 20 mM EDTA and was then phenol extracted and ethanol precipitated in the presence of glycogen and the pellet resuspended in H2O.
It is clear that the procedures described by Mattheakis et al. are very inefficient at capturing and/or recovering mRNA; thus, on p.72 of the Affymax filing (18), only 1-2% of radiolabelled polysomal mRNA encoding the specific peptide epitope was recovered, which was acknowledged to be low (line 5). The patent application (but not the publication) also includes the selection of an antibody fragment, but with much less detail. In this case, Dynal magnetic beads coated with antigen were used as the affinity matrix. In the example, labelled mRNA was specifically recovered but they did not show recovery of cDNA by RT-PCR. Hence there was no estimation of efficiency or sensitivity, and no demonstration of selection from a library or enrichment.
In a more recent publication (15), Hanes and Pluckthun modified the method of Mattheakis et al. for display and selection of single chain antibody fragments. While retaining the concept, additional features were introduced to make the method more suited to display of whole proteins in the prokaryotic, E. coli S30 system. One innovation is the stalling of the ribosome through the absence of a stop codon, which normally signals release of the nascent protein. Once again, recovery of genetic material was by dissociation of the ribosome complexes with 10 mM EDTA and isolation of the mRNA by ethanol precipitation (or Rneasy kit) prior to reverse transcription. Separate transcription and translation steps were used, and it was stated that the coupled procedure has lower efficiency; however, no data was provided to this effect. A large input of mRNA was used in each cycle (10 xcexcg).
Many additions were incorporated by Hanes and Pluckthun in order to improve the yield of mRNA after the polysome display cycle, which was initially as low as 0.001% (15). These included stem loop structures at the 5xe2x80x2 and 3xe2x80x2 ends of the mRNA, vanadyl ribonucleoside complexes as nuclease inhibitor (which also partially inhibit translation), protein disulphide isomerase PDI (which catalyses formation of disulphide bonds) and an anti-sense nucleotide (to inhibit ssrA RNA which in the prokaryotic system otherwises cause the release and degradation of proteins synthesised without a stop codon). The combination of anti-ssrA and PDI improved efficiency by 12-fold overall. However, the yield of mRNA at the end of the cycle, with all additions, was still only 0.2% of input mRNA, expressing the combined efficiency of all steps, including ligand binding (on microtiter wells), RNA release and amplification. Affymax have already described a yield of 2%, i.e. 10-fold higher, as low (cited above).
Hanes and Pluckthun also demonstrated recovery of a specific antibody from a mixture (of two) in which it is initially present at a ratio of 1:108. This required 5 sequential repetitions of the cycle, i.e. using the DNA product of one cycle as the starting point of the next. In FIG. 4(A) of ref. 15, there is a considerable carry over of the nonselected polysomes, probably reflecting the method of selection or mRNA recovery. As a consequence, the enrichment factor is relatively low, about 100-fold per cycle.
A further recent ribosome display method was described by Roberts and Szostak (23), in which the nascent protein is caused to bind covalently to its mRNA through a puromycin link. In this system, selection is carried out on these protein-mRNA fusions after dissociation of the ribosome. It thus differs significantly from the other methods described here since it does not involve selection of protein-ribosome-mRNA particles. Its efficiency is only 20-40 fold.
It is clear that the described prokaryotic methods of polysome display leave considerable scope for methodological improvement to increase efficiency of recovery of mRNA, sensitivity and selection. In the invention described herein, we have developed a novel, eukaryotic method of ribosome display and demonstrate its application to selection and mutation (evolution) of antibodies and to selection of other proteins from mRNA libraries. It could equally be applied to isolation of genes from cDNA libraries.
The invention provides a method of displaying nascent proteins or peptides as complexes with eukaryotic ribosomes and the mRNA encoding the protein or peptide following transcription and translation in vitro, of further selecting complexes carrying a particular nascent protein or peptide by means of binding to a ligand, antigen or antibody, and of subsequently recovering the genetic information encoding the protein or peptide from the selected ribosome complex by reverse transcription and polymerase chain reaction (RT-PCR). The RT-PCR recovery step is carried out directly on the intact ribosome complex, without prior dissociation to release the mRNA, thus contributing to maximal efficiency and sensitivity. The steps of display, selection and recovery can be repeated in consecutive cycles. The method is exemplified using single-chain antibody constructs as antibody-ribosome-mRNA complexes (ARMs). It is suitable for the construction of very large display libraries, e.g. comprising over 1012 complexes, and of efficiently recovering the DNA encoding individual proteins after affinity selection. We provide evidence of highly efficient enrichment, e.g. 104-105-fold per cycle, and examples demonstrating its utility in the display and selection of single chain antibody fragments from libraries, antibody engineering, selection of human antibodies and selection of proteins from mRNA libraries.
In its application to antibody fragments, the method is shown in FIG. 1. In this form, the method is also termed xe2x80x98ARM displayxe2x80x99, since the selection particles consist of antibody-ribosome-mRNA complexes. The antibody is in the form of the single-chain fragment VH/K described above, but the method is in principle equally applicable to any single chain form, such as scFv. The method differs in a number of particulars from those described above, leading to greater than expected improvements in efficiency, sensitivity and enrichment. In principle, it is based on two experimental results: (i) single-chain antibodies are functionally produced in vitro in rabbit reticulocyte lysates (7) and (ii) in the absence of a stop codon, individual nascent proteins remain associated with their corresponding mRNA as stable ternary polypeptide ribosome-mRNA complexes in cell-free systems (8,9). We have applied these findings to a strategy for generating libraries of eukaryotic ARM complexes and have efficiently selected complexes carrying specific combining sites using antigen-coupled magnetic particles. Selection simultaneously captures the relevant genetic information as mRNA.
The coupled transcription/translation system used here is a rabbit reticulocyte extract (Promega) which provides efficient utilisation of DNA. In particular, it avoids the separate isolation of mRNA as described in ref. 15, which is costly in materials and time. The deletion of the stop codon from the encoding DNA is more productive as a means of stalling the ribosome than the use of inhibitors, because it ensures that all mRNA""s are read to the 3xe2x80x2 end, rather than being stopped at random points in the translation process. The stabilising effect of deletion of the stop codon can be explained by the requirement for release factors which recognise the stop codon and normally terminate translation by causing release of the nascent polypeptide chain (26). In the absence of the stop codon, the nascent chain remains bound to the ribosome and the mRNA. Where it is problematic to engineer stop codon deletion as in cDNA or mRNA libraries, an alternative method would be the use of suppressor tRNA (charged with an amino acid) which recognises and reads through the stop codon, thereby preventing the action of release factors (24). A further strategy of ribosome stalling would be the use of suppressor tRNA not charged by an amino acid.
In a novel step which introduces a significant difference from preceding methods, we show that cDNA can be generated and amplified by single-step reverse transcription-polymerase chain reaction (RT-PCR) on the ribosome-bound mRNA, thus avoiding completely the isolation and subsequent recovery of mRNA by procedures that are costly in terms of material and time. The success and efficiency of this step is surprising, since it is generally assumed that during translation several ribosomes attach to the same mRNA molecule, creating a polysome, and it was not known what effect the presence of several ribosomes in tandem on a single mRNA molecule would have on reverse transcription, where the RT enzyme must read the length of the mRNA. Thus, it is not known whether the enzyme might be able to pass through adjacent ribosomes, or cause their removal from the mRNA, or only function on mRNA molecules to which only one ribosome was attached. Whatever the explanation, this step contributes greatly to the demonstrated efficiency of the system, in which up to 60% of the input mRNA can be recovered in one cycle (Example 6, FIG. 9), compared with only 2% in the prokaryotic systems described by Mattheakis et al (14) and 0.2% by Hanes and Pluckthun (15). Furthermore, we have shown that, in the eukaryotic system, extraction of the mRNA from the ribosome complex is five times less effective as a recovery procedure than RT-PCR on the nondisrupted complex and that much of the mRNA remains bound to the ribosome even after EDTA extraction (Example 8, FIG. 11).
The enrichment of individual antibody fragments using ARM display libraries is also more efficient than described for prokaryotic display (15). We have performed experiments which show that mixtures in which the desired specific fragment is present at one part in 105 can yield a binding fragment after one cycle, with an effective enrichment factor of  greater than 104 fold, and that cycles can be run sequentially to isolate rarer molecular species from very large libraries (Examples 10 and 11). This is 2-3 orders of magnitude more efficient per cycle than the results reported in the prokaryotic system (15).
Since the ARM libraries are generated wholly by in vitro techniques (PCR) and do not require bacterial transformation, their size is limited mainly by the numbers of ribosomes which can be brought into the reaction mixture (xcx9c1014 per ml in the rabbit reticulocyte kit, according to manufacturer""s information) and the amount of DNA which can be handled conveniently per reaction. Hence the production of large libraries becomes much easier than in the phage display method, where the limiting factor is bacterial transformation. An important application is in the selection of proteins from large libraries of mutants; the library can be generated through PCR mutation either randomly or in a site-directed fashion and mutants with required specificity selected by antigen-binding. We demonstrate the use of the ARM display procedure to select antibody (VH/K) fragments with altered specificity from such libraries. This application to antibody engineering is shown in Example 12, in which the specificity of an anti-progesterone antibody is altered to testosterone binding by a combination of mutagenesis and selection. Such procedures may also be used to produce catalytic antibodies. The operation of the ARM cycle itself also introduces a low level of random mutation through the errors of PCR and we show that the rate of such errors is 0.54% per cycle (Example 9). This can lead to selection of improved properties of affinity and specificity, and is termed xe2x80x98protein evolutionxe2x80x99 to indicate the development of novel proteins through a combination of mutation and selection (15). The eukaryotic ARM cycle is well suited to carrying out efficient protein evolution in vitro.
The present invention also provides a novel method for obtaining antibodies from libraries made from immunised mice, bypassing hybridoma technology. In particular, we show that it can be used to make human antibodies by employing a combination of transgenic mouse technology and ARM ribosome display. Mice are available in which transgenic loci encoding human heavy and light chain antibody genes are incoporated into the genomes, such mice giving rise to human antibodies when immunised (20). We provide herein an example in which human antibodies are derived in vitro by ARM display of a library prepared from the lymphocytes of such mice (Example 13). This provides a novel route to the derivation of human antibodies for therapeutic purposes.
The ribosome display method described herein is also applicable to any protein or peptide which, having been translated in vitro, remains bound to the ribosome and its encoding mRNA. As well as the examples showing the applicability of ARM display to antibodies, we also demonstrate this more general application through translation of an mRNA library obtained directly from normal tissues for selection of individual polypeptide chains (Example 14).
This version of ribosome display thus meets the need for a simple in vitro display system for proteins or peptides. It is capable of a very large library size, combined with ease and efficiency of selection and recovery of genetic information; it is also less demanding of special conditions, more sensitive and capable of greater levels of enrichment than methods described hitherto. The combination of a eukaryotic system with efficient mRNA recovery provides a system with a far greater efficiency than would have been predicted by those practiced in the art.